UNIVERSITY OF 
-_LINGIS LIBRARY 
Ai URBANA-CHAMPAIGN 
BOOKSTACKS 


Digitized by the Internet Archive 
in 2013 


http://archive.org/details/effectoflightongOOgard 


The University of Chicago 


EFFECT OF LIGHT ON GERMINATION 
OF LIGHT-SENSITIVE SEEDS 


A DISSERTATION > 


SUBMITTED TO THE FACULTY 
OF THE OGDEN GRADUATE SCHOOL OF SCIENCE 
IN CANDIDACY FOR THE DEGREE OF 
DOCTOR OF PHILOSOPHY 


DEPARTMENT OF BOTANY 
@e- * 


BY 
WRIGHT AUSTIN GARDNER 


Private Edition, Distributed By 
THE UNIVERSITY OF CHICAGO LIBRARIES 
CHICAGO, ILLINOIS . 
_ Reprinted from 
THE BOTANICAL GazETTE, Vol. LXXI, No. 4, April 1921 


before the 
Return this book on s below. 


Latest Date stampe 


ing of books ’e) 
Theft, mutilation, and angie a aha 


MAR 2 0 1976 


Eon 4 MINATION 
_warii2 275 =O EEDS 


BH {8bb 


No 27h MAR lo ‘#70 
wv og 
pcr SUIS gee DI * 


BR Mie 

> Mr le 
MAR - 311874 
FEB 25 197 


L161— 0-1096 


Private Edition, Distributed By 
THE UNIVERSITY OF CHICAGO LIBRARIES 
CHICAGO, ILLINOIS 


Reprinted from 
THE Botanica Gazette, Vol. LXXI, No. 4, April 1921 


The University of Chicago 


EFFECT OF LIGHT ON GERMINATION 
OF LIGHT-SENSITIVE SEEDS 


A DISSERTATION 


SUBMITTED TO THE FACULTY 
OF THE OGDEN GRADUATE SCHOOL OF SCIENCE 
IN CANDIDACY FOR THE DEGREE OF 
DOCTOR OF PHILOSOPHY 


DEPARTMENT OF BOTANY 


BY : 
WRIGHT AUSTIN GARDNER | 


Private Edition, Distributed By 
THE UNIVERSITY OF CHICAGO LIBRARIES 
CHICAGO, ILLINOIS 


Reprinted from 
THE BoTANICAL GAzETTE, Vol. LXXTI, No. 4, April 1921 


VOLUME LXXxXI NUMBER 4 


eh 
MEOOTANICAL (GAZETTE 
PILE TO2T 


EFFECT OF LIGHT ON GERMINATION OF 
LIGHT-SENSITIVE SEEDS 


CONTRIBUTIONS FROM THE HULL BOTANICAL LABORATORY 279 - 


Wricut A. GARDNER 
Historical 


Various explanations have been offered for the germination of 
light-sensitive seeds, and several conditions have been shown to 
favor or make possible the germination of such seeds. Rupture 
of coats, increased water supply, variation of quantity and intensity 
of light, reciprocal relation of heat and light, reaction of substratum 
and embryo, activation of enzymes, increased oxygen pressure, 
increased carbon dioxide pressure, and “certain inhibiting agencies”’ 
have been suggésted as factors affecting the germination of light- 
sensitive seeds. Although quite possible, it seems hardly probable 
that no one of these is the fundamental or controlling factor. It 
would seem quite probable that one or two of these agencies are 
fundamental and the others are accessory means of setting in 
motion the processes that finally bring about germination. Enzyme 
action has been suggested repeatedly as a fundamental cause of 
germination, but no one has ventured to demonstrate the relation 
of enzymes to the germination of light-sensitive seeds. 

An attempt has been made in this investigation to discover the 
fundamental relation of light to the germination of seeds, and to 
show just what light does to start germination. The effect of light 
on the germination of seeds has interested botanists for many years. | 


249 


250 BOTANICAL GAZETTE [APRIL 


The first known publications on this subject were made by CASPARY 
(4) in 1860, when he announced that the seeds of Bulliarda aquatica 
are strongly light sensitive. In 1867 he (5) discussed the germina- 
tion of seeds of Pinguicula vulgaris. In 1876 Nope (38) made 
the statement that germination was neither favored nor influenced 
by light. After WIESNER (51) had published the statement that 
the germination of seeds of Viscum album is favored by light, and 
STEBLER (48) had shown that Poa pratensis and P. nemoralis 
germinated up to 60 per cent in light and only up to 7 per cent in 
darkness, NOBBE (39) published results of experiments with grass 
seeds, including Poa pratensis, Zea Mays, and some other large 
seeds to uphold his earlier contention. PAucHON’s (41) results 
supported NosBE in the controversy. In 1883 CIESLAR (6) con- 
firmed and extended STEBLER’s results, reporting Agrostis stolonifera 
and Nicotiana macrophylla as light sensitive. He made a careful 
study of the influence of temperature in connection with light, and 
showed that small seeds poor in reserve materials germinate better 
in white light, while large seeds are usually indifferent to light, and 
that seeds of Poa nemoralis germinate better in yellow light than in 
violet. LIEBENBERG (35) in 1894 confirmed STEBLER’s results, 
but referred to them as temperature effects. 

In 1893 JONSSON (23) showed that after-ripening has a definite 
influence on the action of light in germination, that light increases 
the percentage of germination, that heat rays are unimportant, 
that intermittent light is as effective as continuous light, and that 
intermittent temperature may be substituted for light in the 
germination of such seeds as Poa pratensis, P. nemoralis, Agrostis 
stolonifera, and Daucus Carota. 

In 1899 HEINRICHER (19) began publishing the results of his 
work on light germination. He (20) reported that seeds of Pit- 
cairnia maydifolia germinate only in light, that the germination 
of Veronica peregrina seeds is hastened and several other small seeds 
are favored in germination by light. He considered the factors to 
be age, quickness of drying, moisture, illumination of parent plant, 
and light of different colors. He (21) concluded that the effect 
of light is a matter of activation of reserve materials, that the 
benefit of light is not due to its causing early carbon assimilation, 


1921] GARDNER—GERMINATION 251 


but rather to its effect on the enzyme activity in the production 
and digestion of stored foods. RAcrtBorskt (43) found that tobacco 
seeds germinate in diffused light after 1-5 hours’ illumination, a 
longer time being required if the intensity of light is low. In 1900 
TamMes (49) declared that the exposure of dry seeds to direct 
sunlight did not affect their later germination, and in 1902 LAURENT 
(29) made the same statement. REMER (44) reported that light 
hinders the germination of Phacelia tanacetifolia, but offered no 
explanation of the light relations. LascHKE (28) confirmed earlier 
results with Poa, and stated that light cannot be replaced by 
higher temperatures. In the same year Ficpor (10) made a report 
on the influence of light on the germination of seeds of Gesneriaceae. 
In 1912 he (11) reported that seeds of 12 species of this family are 
favored by light. In 1908 BrEssEy (3) found that seeds of the | 
epiphytic Ficus aurea and F. populnea germinate only in light. 
KiNZEL (24) in 1907 reported that the germination of freshly 
harvested seeds of Nigella sativa was prevented by light, while 
similar seed germinated up to 94 per cent in darkness. Even three 
minutes’ illumination after 24 hours’ incubation in darkness had a 
marked retarding effect. He considered the effect of light as photo- 
chemical, and designated such seeds as “light hard.”’ The germina- 
tion of some light-sensitive seeds in darkness was hastened by 
soaking in a solution of an enzyme such as papayotin (25). He 
(26) also published a long list of light-sensitive seeds, to which still 
others were added in his later work (27). He recognized as 
important factors in germination of seeds age of seed, character of 
seed coats, and color of light. LEHMANN’s work (30, 31, 32; 33) 34), 
begun in 1909, continued through 1915. Most of his experiments 
were conducted with seed of Verbascum Thapsus, V. thapsiforme, 
Epilobium roseum, and Ranunculus sceleratus. He showed the 
effects of substratum on germination in light, found the age of 
seeds to be an important factor, used Knop’s nutrient solution as 
a stimulus instead of light, and found that salts favored germination 
of light-sensitive seeds in darkness. He claimed that light exerts 
its influence by starting or stopping some chemical changes in the 
seed, and established a relation between light and temperature. 
He also punctured seed coats as a substitute for light. LEHMANN 


2G BOTANICAL GAZETTE [APRIL 


and OTTENWALDER (36) experimented with Epilobium lursutum 
and other seeds and showed that acid solutions and proteolytic 
enzymes can be substituted for light. They referred the light 
effect to activation of enzymes, but did little to prove their 
hypothesis. PicKHoLtz (42) connected light effects and tempera- 
ture variations, and concluded that the influence of direct sunlight 
was mainly due to the heat rays which raised the temperature. 
Alternating temperatures helped the germination at different stages 
of maturity. 

In 1912 a number of workers reported on the problem of light 
germination. BAar (1) investigated seeds of several Amarantaceae 
and found that most seeds of this family (Amaranthus, Celosia, and 
Blitum) have an aversion to light. He considered the age of seeds 
generally important for the occurrence and intensity of the light 
effect, and also related the light effect to substratum and temper- 
ature variations. BECKER (2) brought forward a long list of 
examples of the light effect on germination of seeds. Haack (18) 
in his work on the Scotch pines demonstrated the influence of 
heat, and reported that temperature variations act as a stimulus to 
light-sensitive seeds, and that blue light is more favorable to germi- 
nation than darkness. Simon (47) reported that the salts of iron 
hindered germination of seeds in darkness, but increased it in light. 
GASSNER (12) first reported on the germination of seeds of Chloris 
ciliata in 1910. He found three factors which may be substituted 
for light, namely, increased oxygen supply, after-ripening, and 
high temperature. He claimed that light offsets the effect of the 
limiting factor, and showed that the chaff of Chloris ciliata prevents 
easy germination. His later work (14, 15) took up the action of 
chemicals. He considered the latent influence of light as related 
to seed bed, temperature, and after-ripening, the influence of light 
on germination, the influence of desiccation, and the relations 
between light and media favoring or hindering germination. From 
a tabulation of tests with seeds of different families he concluded 
that in these cases nitrogen, variously combined in the media, 
shows the same favorable action as light, but he included contradic- 
tory results. He considered the favorable effect of Knop’s nutrient 
solution as due only to the nitrates present. He reported the seeds 


1921] GARDNER—GERMINATION 253 


of Ranunculus sceleratus and Oenothera biennis as favorably influ- 
enced by light and by inorganic salts containing nitrogen, through a 
wide range of concentrations. OTTENWALDER (40), working with 
Epilobium hirsutum seeds, found that the light requirement as 
regards intensity is closely related to temperature, the former 
increasing as the latter is lowered. The illumination period is 
related also to the temperature, but more closely to light intensity. 
Light-sensitive seeds are also favorably and strongly influenced 
by weak acids. The hypothesis of a catalytic influence of light 
is said to have received support from these observations. 


Materials 


A preliminary examination of 115 samples of seeds collected 
from Shaw’s Gardens, the Botanical Gardens of the University of 
Michigan, waysides, swamps, and fields indicated the following as 
available for the study of the effect of light in germination: Daucus 
Carota, Nicotiana Tabacum, N. sylvestris, N. affinis, Nicotiana 
hybrids, Gentiana Saponaria, G. pannonica, Oenothera biennis, 
Verbascum Thapsus, Amaranthus caudatus, Rumex crispus, Phora- 
dendron flavescens, and Datura Stramonium. Of those mentioned, 
the writer has been unable to find any previous report of light 
sensitiveness of seeds of Rumex crispus, Datura Stramonium, and 
Phoradendron flavescens. Oi the light-sensitive seeds not previ- 
ously reported, séeds of Rumex crispus and Phoradendron flavescens 
are favored by light in germination, while seeds of Datura Stra- 
monium are inhibited from germinating by light, as will be shown 
later. 

JONSSON (23) in 1893 reported the seed of Daucus Carota as 
favored by light in germination. Nicotiana Tabacum seeds were 
first reported as light sensitive by RAcIBoRSKI (43) in 1900. The 
seeds of Gentiana Saponaria, G. pannonica, Verbascum Thapsus, 
and Oenothera biennis were reported as light sensitive by KINZEL 
(24) in 1907. BAAR (1) reported Amaranthus caudatus seeds as 
hindered in germination by light. The seeds of Gentiana are not 
conveniently suited to the purposes of this investigation on account 
of the longer incubation period. The seed of Amaranthus caudatus 
are not used because they are light-inhibited seeds. The seeds 


254 BOTANICAL GAZETTE [APRIL 


of Datura Stramonium have been found by careful experiments to 
be light inhibited and to require total darkness and a temperature 
of about 30°C. for germination. They are accordingly reserved 
for a future study. Detailed study of the germination of Phora- 
dendron flavescens seeds was deferred on account of the peculiar 
slimy ovary and the chlorophyll-bearing embryo. Seeds of Nico- 
tiana Tabacum, Rumex crispus, Oenothera biennis, Verbascum Thap- 
sus, and Daucus Carota were selected for this research because of 
their abundance and similar incubation periods. 


Germination in light and darkness 
| oranda tests of Rumex crispus seeds on wet filter paper 
gave a germination of 84 per cent in light and 16 oes cent in dark- 
ness after 8 days of incubation. 


TABLE I 
Percentage Percentage 
Treatment germination in | germination 
light in darkness 
Cleaned, dried, and soaked in 
flowing water for 24 hours 
Ang IncWloatedenan eee eee 16 6 
Cleaned, dried, and incubated 24 6 
Cleaned, soaked for 4 days, and 
MG ate is he ey pine we 12 2 


Preliminary tests of germination of seeds of Phoradendron 
flavescens, suggested by WIESNER’S (51) results with seeds of 
Viscum album, are given in table I. Seeds prepared as indicated in 
table I were counted into Petri dishes containing wet filter paper 
as substratum and placed in light and total darkness to incubate 
at room temperature, which ranged from 18-25°C. The incubation 
period was 27 days. These results indicate that light favors the 
germination of seeds of Phoradendron flavescens. On account of 
the sticky nature of the pulpy ovary and the succulence of the single 
fleshy green embryo, it was almost impossible to remove the mass 
of enveloping material without leaving a favorable substratum for 
‘molds and bacteria on the one hand, and without injury to the 
embryo on the other hand. Moreover, with the best of care many 
of the seeds failed to germinate and became moldy. With these 


1921] GARDNER—GERMINATION 255 


conditions we can understand the relatively low germination, and 
yet see that light favors the germination of these seeds. 

Seeds of Datura Stramonium were treated as shown in table II. 
Seeds were allowed to incubate for 17 days, and the results indicate 
clearly an inhibitory action of light on their germination. ‘The 


TABLE II 
| Percentage | Percentage 
Treatment germination | germination in 
in light darkness 
ON CSOU HOG 280 WG eo sess rk, ole 6 98 


Onrsand 20-20 fou. cs. so 2 22 

Onilter papery 26 -C oo svaen ss fe) fo) 

On milter paper, 240-C i. 1... fo) 4 
4 


On filter paper, 30°C... cnc a - 60 


constituents of the soil solution seem to promote materially the 
germination of these seeds in darkness but not in light. Datura 
Stramonium seeds require different treatment from any of the other 
seeds tested, and accordingly are reserved for separate study. 


Light sensitiveness, after-ripening, and viability 


To establish standards for comparison with other data, and to 
indicate the degree of light sensitiveness, various light-sensitive 
seeds were incubated from time to time at room temperature 
(20-28° C.) on filter paper in light and darkness respectively. The 
results given in table III are fairly representative of these tests. 

These data indicate what may be expected of the various light- 
sensitive seeds under investigation when subjected to germinating 
conditions at room temperature 20-28° C. on wet filter paper in 
Petri dishes. It appears that the seeds of some kinds of tobacco 
are less light favored than others (3. Nzcotiana affinis, 108. N. affinis, 
1. Nicotiana hybrid, and 117. Pennsylvania Havana tobacco). 
The results also indicate that not all of the seeds under investigation 
are entirely dependent on light for germination. A certain per- 
centage of each lot of Rumex crispus and Daucus Carota seeds 
usually germinates in darkness. It is also noteworthy that seeds 
of Oenothera biennis do not always germinate, even in light. The 
seeds under investigation seem to retain their light sensitiveness 


256 BOTANICAL GAZETTE [APRIL 


for long periods and to a rather high degree, especially those of 
Verbascum Thapsus and Nicotiana Tabacum. Attention should be 
called to low germination of newly harvested seeds of Oenothera 
biennis, Daucus Carota, and Rumex crispus. ‘Tests for evidence of 


TABLE III 
| INCUBATED INCUBATED INCUBATED INCUBATED 
9-18-15 TO Io-28-15 TO 5-30-16 TO 6-20-18 TO 
9-20-15 LI-S=15 6-6-16 6-28-18 
SEEDS 
ves | Bake nike | Pat | em | Bat | nih | Bae 
Collected in 1914 

1. Nicotiana hybrid...} 91 56 87 65 87 60 84 45 
3. Nicotiana affinis....| 79 Sy 87 45 74 43 82 19 
8. Nicotiana hybrid....| 48 f 58 5 74 35 67 13 
13. Nicotiana hybrid....| 84 fo) 89 2 89 6 80 ° 
22. Nicotiana hybrid....| 55 ) 73 ° 61 ° 55 fe) 
55. Verbascum Thapsus.| 81 I 80 ° 87 ° ee fe) 
61, Daucus Carotay. 7% 63 19 78 10 7a 20 32 7 
66. Oenothera biennis.. . 4 2 76 10 pi 4 ) ° 
68. Rumex crispus...-2--| “36 8 88 18 75 Si 60 i 
92. Nicotiana Tabacum.| 77 NT eee ee dn Cee a go 4 30 ° 

96. Daucus Carota..... I fo) 52 6 |......|.... 290 


Collected in 1915 


97. Verbascum Thapsus.| 94 fe) 94 ° 82 o 4) Ae 
98. Oenothera biennis.. . 2 fo) 48 fo) 61 I | ng 
og. Rumex crispus...... 36 fo) 82 4 80 40 66 ° 
100. Verbascum Thapsus.| 74 I 73 ° 39 fo) 60 fo) 
tor, Rumex crispus;......| “6a fo) 04 fo) 92 2 30 fe) 
102: Daweus Catota. +. sole. tee 45 I 44 tr ||. (2S eee 
103. Oentotherabienmig;o |igancthekn <2 63 fe) 61 © ||. #6) Bee 
10/4. JRUMEX CFISPUS =. a a2). Poe od Se Fale 94 I 79 Il |. .3-s eee 
105. Pennsylvania seed- 
leat £6 baCCOse ak Se Beat ee ee ee 75 24. |i cee 
111. Connecticut seedleaf 
LOWACCOL Lis. Seer Os aie dete exalt ete ene 75 Oo |. =a 
T12.Hondutas LObACCOIe |< Ge tretce eon eee eee 49 © |. +,aeeeee 
Tra, Cuban tobaccove cs iy. ey fae tess eee ee ee 81 o |. eae 
117. Pennsylvania Ha- 
VANALOMACCO fers alte s. Gils 2 oe pale eee ee 100 7t |e se 
176: 'Ohioseadleattobaccolel aa Ahi eels ed eee 86 2 | .. ea 


a period of after-ripening in Verbascum Thapsus were quite nega- 
tive. Newly harvested seeds of V. Thapsus germinate above go 
per cent in light and only about 2 per cent in darkness. Tests of 
still older seeds indicate that they retain light sensitiveness as long 
as they are viable. 


1921] GARDNER—GERMINATION 257 


Mechanical rupture 


In 1906 CROCKER (7) succeeded in germinating a number of 
different kinds of seeds after breaking the seed coats. KINZEL (24) 
found that puncturing coats of some of his light sensitive seeds 
gave better germination in darkness. GASSNER (13) found that 
rupture of the coats of seeds of Chloris ciliata permitted good 
germination in darkness at 34°C. ‘Thus it seemed quite possible 
that the seeds under investigation might be brought to germination 
by such treatment. Accordingly a more carefully controlled 
experiment was made to determine the rdéle of the several seed 
coats in germination. Seeds of each kind were rubbed on fine 
sandpaper and placed on moist filter paper in Petri dishes. The 
Petri dishes were carefully wrapped in black cloth and placed in 
a dark room at 24-30°C. for 8 days. Concurrently, sets of. 
unabraded seeds were placed to germinate in light and darkness. 


TABLE IV 
Not 
NoT ABRADED ABRADED ek GeS 
SEEDS elt See 

Light Darkness | Darkness | Darkness 

Miemttana Vabacum................... 60, 61 0, O One o, I 
Ruimex Coispus......... 2 Be ae ae 30, 16 Gy. E 40, 66* Gr. 
Weetascem Paapsus............... ee ale 5a 32 G..8 Gin Tx 2 
MipieemeeeNMIS, .. 18... ke 10, 39 2,0 Zuni anal 
Daneus Carota...:..... 2 a es 60, 20 a a 25 0s CT U7) 


* Coats off. 


Mechanical abrasion of seed coats for various periods in rotating 
cylinders containing coarse quartz sand gave similar results. An 
examination of the data in table IV reveals the beneficial effect of 
abrasion of seed coats in but one instance. In the case of Rumex 
crispus abrasion of the seed coats yielded a percentage of germina- 
tion slightly exceeding that for light in the control, while the removal 
of the coats yields a percentage of germination even more than 
double that in light. This suggests that the seed coats of Rumex 
crispus inhibit or retard the entrance of some necessary factor, 
or perhaps retard the exit of some inhibiting factor, and that light 
in some way favors these movements. 


258 BOTANICAL GAZETTE [APRIL 


Rupture by sulphuric acid 


As long ago as 1896 Rostrup (45) of the Danish Seed Con- 
trol found that concentrated sulphuric acid treatment hastened 
germination of hard seeds of Lathyrus sylvestris. TODARO (50) 
used concentrated sulphuric acid on red clover seed with beneficial 
results. He also reported that various weed seeds, including those 
of Rumex crispus, were all destroyed by a brief immersion in 
sulphuric acid. Accordingly, to determine more certainly the réle 
of seed coats in the germination of the five kinds of seeds, they 
were treated with concentrated sulphuric acid for periods previously 
determined, carefully washed in carbonate of soda solution, then 
in distilled water, and placed in germinators as previously described. 


TABLE V 
TREATED WITH CONCENTRATED SULPHURIC ACID UNTREATED 
earns ‘ : Germinated in darkness ; 
Minutes in In light |Indarkness 
2590, 8 days 8 days 


8 days Io days 8 days 
(z) (2) (3) 


Nicotiana Tabacum....... 


0.5 fo) ° fo) 42 fo) 
Nicotiana Tabacum... 7... I ) ° ° 42 ° 
Verbascum Thapsus...... 0.5 O° fo) fe) 72 fe) 
Verbascum Thapsus...... I fo) fe) fe) 72 fe) 
Dancus Catotaie a. ics 248 0.5 23 8 Oo Se 25 
Daucus Carota: te. : aoe I I 6 fe) ae 25 
IRGHHEX ChISPUS 2 en's te, 3 oe. 6 27 a7 17 88 ° 
RiGnex Crispus... 264s 8 62 59 ° 88 ° 
Oenothera biennis........ 8 38 34 4I 78 ° 
Oenothera biennis........ ime) I 19 23 78 ° 


Table V indicates that treatment of seeds of Rumex crispus and 
Oenothera biennis with concentrated sulphuric acid yields an 
increased percentage of germination in darkness. Treatment with 
concentrated sulphuric acid for longer or shorter periods than 
‘indicated gives no better germination of the seeds in darkness. In 
Daucus Carota there is apparently an injury. This experiment 
indicates that light acts on the coat of Rumex crispus seeds, and 
points in that direction in case of seed coats of Oenothera biennis. 
‘These results in the main agree with those of the experiment on 
abrasion of coats. They confirm the results with seeds of Rumex 


1921] GARDNER—GERMINATION 259 


crispus and include the seeds of Oenothera biennis as being benefited 
in germination by acid treatment. Why the seeds of Oenothera 
biennis germinate better after treatment with H,SO, and not by 
abrasion is unexplained. 


Temperature and light 


OTTENWALDER (40) claimed that within the range of tempera- 
tures which permit germination, light can be substituted for heat 
at low temperatures and heat for light at high temperatures. With 
Verbascum Thapsus and other seeds he found that germination 
occurred at high temperature in darkness and at low temperature 


TABLE VI 


Temperature centigrade 


Seeds 20°. | Ego | r9° | 24 


27 
Light 
Nicotiana Tabacum...... fo) ° | 23 62 55 SG he | RE 
Verbascum Thapsus..... ° eae 77 82 oy, a Pe ad Wer a 
Pimess Catota.. ........ fo) 12 21 36 33 DE is RO a Pe eee 
Oenothera biennis....... O° [ 6 pee Werte es ot Neer aee Visa. 
Rumex crispus.......... ° | Bo ae 65 BON I me Bi yt? slo Saupe 
| 
Darkness 
Nicotiana Tabacum...... ° ° ° II 6 2 fo) fe) 
Verbascum Thapsus..... ° ° ° 7 14 ras 3 I 
Daucus Carota........ he ° 4 7 16 12 12 4 fo) 
Oenothera biennis....... fo) ) fe) 5 4 2 ° ° 
amex crspus. ......... ° I 3 8 Se, le ° | fe) 


in light. In order to test this for American Verbascum Thapsus 
- and to see whether it is generally true, the different seeds under 
investigation were placed to germinate in light and darkness at 
different temperatures. It was not possible to control closely 
temperature and prevent small fluctuations. These changes of 
temperature were never sudden, however, and had no effect except 
to increase slightly the percentage of germination in both light and 
darkness. The data reported represent results obtained from five 
different sets of determinations. Seeds were placed to germinate in 
Petri dishes on filter paper wetted with distilled water at tempera- 
tures indicated in table VI and allowed to incubate for 9 days. 


260 BOTANICAL GAZETTE [APRIL 


These data offer no evidence of a reciprocal relation between 
heat and light as suggested by LEHMANN and OTTENWALDER, not 
even in the case of seeds of Verbascum Thapsus, nor have any of 
the various tests indicated this reciprocal relation in the seeds. 
Indeed, in each kind of seed under investigation the optimum 
temperature for germination in light is very close to that for 
germination in darkness. In germination in darkness the results 
show rather definite minimum and maximum as well as optimum 
temperatures. It is especially noteworthy that high temperature 
and darkness did not induce germination of Verbascum Thapsus 
seeds, as claimed by OTTENWALDER (40). No specific experiments 
were performed to determine the effect of light intensity on germina- 
tion, although early in-this investigation it became a very familiar 
fact that very little illumination would induce germination. Good 
germination in darkness was frequently the occasion for repetition 
of an experiment, only to find that germination had been. induced 
by leaks in the light screens. A comparison of table VI with data 
given elsewhere indicates that highest germination of light-sensitive 
seeds does not occur at constant temperature, but at temperatures 
fluctuating between 20 and 27°C. 


Effects of alternation of temperature, light, and darkness 


As long ago as 1882 NoBBE (39) and his students used alternating 
temperatures to promote germination, and in 1884 LIEBENBERG (35) 
referred light effects to variations of temperature in the germination 
of seeds of Poa pratensis. As recently as 1911 PICKHOLTZ (42) 
referred the action of light in promoting germination to the 
effects of heat rays. In an attempt to distinguish the effects of 
light from those of temperature the following experiments were 
performed. Seeds of each kind were counted into Petri dishes with 
filter paper wetted with distilled water as substratum. One lot of 
cultures was placed in darkness on February 9g at 40° C., where it 
remained for 17 days. Another lot of cultures was placed in dark- 
ness at temperatures ranging from o to 12° C. for 17 days. Another 
lot was kept in darkness and subjected alternately to high tempera- 
ture (40°C.) and low temperature (o-12°C.) for nearly equal 
periods throughout the 17 days. The low temperature and the 


1921] ’ GARDNER—GERMINATION 201 


alternating temperature cultures were frozen on the morning of 
February 26. On this date observations were made and the cultures 
placed in light at room temperature to test viability. 

As shown in table VII, the constant high temperature effectively 
inhibited the germination of all seeds except those of Verbascum 
Thapsus. ‘The subsequent incubation in light at room temperature 
showed fatal injury to the embryos of Daucus Carota, Oenothera 
biennis, and Nicottana Tabacum. The constant low temperature 
delayed germination, but seemed to induce increased germination 
in light in seeds of Daucus Carota, Oenothera biennis, and Verbascum 
Thapsus. This is especially noticeable in Oenothera biennis seeds. 


TABLE VII 


ALTERNATELY AT 

40 AND o-12°C. 

IN DARKNESS FOR| CONTROL AT 
I7 DAYS (@) AND |] ROOM TEMPERA- 


CONSTANTLY AT | CONSTANTLY AT 
40° C. IN DARK- |o-12° C. IN DARK- 
NESS FOR 17 DAYS|NESS FOR 17 DAYS 
(@) AND THEN AT | (@) AND THEN AT 


THEN AT ROOM TURE FOR II 
SEEDS ROOM TEMPERA- | ROOM TEMPERA- TEMPERATURE DAYS 


TURE IN LIGHT | TURE IN LIGHT 
FOR 12 DAYS (b) | FOR 12 Days (0) ST eC eS 


I2 DAYS (bd) 
ee TONES Gale |G) ata): 9.) | tignt | Date 
Verbascum Thapsus..... 14 78 fo) 86 6 fare) 76 2 
IMEX CrISPUS.... 62%... ° 54 KS) 58 ° 56 82 30 
Wamcws Carotas..0..... fo) I ° 60 fo) Oi le ee 6 
Oenothera biennis....... fo) 4 fo) 64 ° go 14 fo) 
Nicotiana Tabacum......| 0 ° ° 40 fo) 26 58 fe) 


The alternating high and low temperature treatment delayed the 
germination in the same way as did constant low temperature. 
As shown by the subsequent incubation, Daucus Carota seeds were 
injured most. The germination of seeds of Nicotiana Tabacum 
and Rumex crispus was materially reduced by this treatment, while 
the germination of seeds of Verbascum Thapsus was favored, and 
the germination of seeds of Oenothera biennis very greatly increased. 

In a further effort to distinguish effects of light and temperature 
an experiment was carried out as follows. Seeds were counted 
into Petri dishes, having filter paper wetted with distilled water for 
substratum, and placed under the following conditions: set a in 
light at 10° C. for 8 days and then at 25°C. for 8 days; set 6 in 
darkness at 10° C. for 8 days and then at 25°C. for 8 days; set c 


262 BOTANICAL GAZETTE [APRIL 


in light at 10° C. for 8 days and then in darkness at 25° C. for 8 days; 
set d in darkness at 1o° C. for 8 days, then! at 40° C. iormideye 
followed by 4 days at 25°C. At the end of the 16 days’ treatment 
all of the cultures were placed in light for 8 days at room tempera- 
ture. The experiment was begun July 24. 

Comparison of the data in a of table VIII with the control 
indicates that incubation in light at low temperature followed by 
incubation at room temperature results in reduction of percentage 
of germination of Daucus Carota seed. A comparison of a and 
the control indicates that alternating temperatures may in a 
measure replace light in the case of germination of Verbascum 


TABLE VIII 
S b 
ue (a) (b) (c) (d) 

BO w . » 

4.03 J oO 0 5 » O ©) = 

A aA fe a uw os ra SA a « a SA ° a Si = 

gez io |S |2 (2/2 (2 ib |gs|s fe eed 

SEEDS 2) 5 NF ee a ie be Sm [eco ie a ets 
2 le, |e jasie |Sslt [es les lesoesles 
Aa RS a a |Sel a Melis | GS] Mae | Galt | oa 
4 (S212) ye (es) 22 | So 1s wl | Soe 
2 (S8| 82] 8S |wol ae | Soo |S 8] BO | Goo | fools GO| Goo 
s aq | WO) av ad |seul gd Be LOO) so) oe 1 Broo! ot 
so | g |G] 20 | B00 = Bo | 2S 70] 20 2a Sie +8 £2 

we | A lA |e BH ie | & H la |e BH iS |e a 
VerbascumThapsus| 94 | 6 |:0 | 95) | 98 | ©.| 64 | 83 | © | 7a | Vauieeee 
Rumex crispus.. 4) S0)|.2)|eOu|eQo0)|"05, 40 2.| 63 |-© |:O4 | OF | @ leaaeeee 
Daucus Carota.....| 6074 \foumadar 15°] 1o 2| 16 | 0 | IE | Fa) oO eee 
Oenothera biennis.| 85 | 0 | o | 69 | 69 | o | 16 | 16] o 2 2) ©) A wate 
Nicotiana Tabacum| 53 | 0.0} 70 | 7210] of 3 | ©} 35) gone I 


Thapsus seeds, that it is an important factor in the germination of 
Rumex crispus seeds, and further indicates the necessity of light 
in the early periods of incubation of Daucus Carota, Oenothera 
biennis, and Nicotiana Tabacum. A comparison of 0 with a points 
again to the necessity of light in Rumex crispus, Daucus Carota, 
Oenothera biennis, and Nicotiana Tabacum, and indicates that 
some inhibiting factor developed during the 8 days in darkness in 
the case of Oenothera biennis and Nicotiana Tabacum. A com- 
parison of c with the control indicates that light does its work on 
such seeds as Verbascum Thapsus, Rumex crispus, and in a measure 
on Nicotiana Tabacum even at low temperature, and that as soon 
as heat is supplied germination occurs. Incubating Daucus Carota 
and. Oenothera biennis seeds at low temperature for a period of 8 


1921] GARDNER—GERMINATION 263 


days, in light or darkness, produces a condition from which they 
do not recover when incubated at 25° C. in light or in darkness. A 
comparison of d with control a, 0, and c indicates that sudden 
changes from extremes of temperature may delay germination of 
Verbascum Thapsus seeds, that such treatment inhibits the germina- 
tion of a large percentage of Rumex crispus seeds, and that it almost 
entirely inhibits the germination of seeds of Nicotiana Tabacum. 
The results in d confirm the observations on Daucus Carota and 
Oenothera biennis made in connection with 6, namely, that some 
limiting factor develops during incubation in darkness at low 
temperature which is not easily overcome. The most noteworthy 
result of this treatment is the complete inhibition of germination 
of seeds of Nicotiana Tabacum. This isin agreement with that found 
in b. Together these results when compared with the control indi- | 
cate a light requirement for Nicotiana Tabacum seeds which is not 
replaced by any temperature combination tried. 

To summarize, this experiment shows that alternating tempera- 
ture may replace light in germination of Verbascum Thapsus seeds, 
that light is necessary for optimum germination of entire seeds of 
Rumex crispus, although change of temperature in a measure 
replaces light. The results of this experiment indicate that seeds 
of Oenothera biennis and Daucus Carota require light and medium 
temperature for optimum germination, and that incubation at low 
temperature in darkness permits a change which is not overcome 
by transfer to high temperature in darkness. Moreover, in Daucus 
Carota exposures to light at 25° C. did not bring about germination 
of these changed seeds. Incubation of Nicotiana Tabacum in 
darkness at to° C. did not result in increased percentage of germina- 
tion in darkness. Incubation of Ncotiana Tabacum seeds in light 
at 10° C. promoted subsequent germination in darkness. 


Hot water treatment 


In a further attempt to induce germination in darkness, an’ 
adaptation of the warm bath method of Mo.iscH (37) was 
employed. The seeds were counted, wrapped in filter paper, 
inclosed in little bags of cheesecloth, and plunged into hot distilled 
water for 0.25 minute and o.5 minute respectively. Great care 


264 BOTANICAL GAZETTE [APRIL 


was taken to plunge them promptly into cold distilled water, when 
the hot water was squeezed out. The seeds were then placed to - 
germinate for 7 days at room temperature under the usual con- 
ditions. Table IX indicates what may be expected of hot water 
treatment of seeds. Treatment at lower temperatures was inef- 
fective and so was not tabulated. The experiment was begun 
March 4. 

The results of the warm bath treatment are mostly negative. 
The percentage of germination of Rumex crispus in darkness is 


TABLE IX 
2g TREATMENT AT | TREATMENT AT | TREATMENT AT UNTREATED; 
&& |90°C.; GERMINA-|75°C.; GERMINA-|60° C.; GERMINA-| GERMINATION 
= : TION AT ROOM TION AT ROOM TION AT ROOM AT ROOM 
S = TEMPERATURE TEMPERATURE TEMPERATURE TEMPERATURE 
SEEDS = S FOR 7 DAYS FOR 7 DAYS FOR 7 DAYS FOR 7 DAYS 
ge 
E ie Light as Light po Light poe Light pr 
NicotianaTabacum| o. 25 4 2 ° fo) 42 ce 62 2 
NicotianaTabacum] 0.5 ° ° ° ° 50 34. ep see 
Daucus Carota...| 0.25 22 fe) 24 6 20 i 52 2 
Daucus Carota...| 0.5 22 Oo 36 8 40 8 |i. a eel eee 
VerbascumThapsus| 0. 25 ° ° 75 ie) 67 20 78 fo) 

. VerbascumThapsus| o. 5 ° ° 75 8 65 5 12% ode gee 
Oenothera biennis.| 0. 25 70 20 76 20 40 8 50 6 
Oenothera biennis.} 0.5 ae: 6 80 48 28 ‘20. ||)? a ee 
Rumex crispus....| 0.25 68 4O 34 12 58 35 66 18 
Rumex crispus....| 0.5 66 36 58 IO 86 28. ly age ee 


increased somewhat by treatment with hot water at 90° C., while 
that of Oenothera biennis is increased somewhat by treatment with 
hot water at 75° C. and go° C. These results indicate the coat as the 
limiting factor in their germination. ‘Treatment at 100° C. for short 
periods might furnish interesting information. 


Water absorption 


To determine the relation of water absorption to germination 
in light and darkness, 2 to 3 gm. of each of the different kinds of 
seeds were weighed separately and placed under favorable con- 
ditions for germination. As soon as the first germination in light 
was observed, the seeds were dried rapidly and weighed carefully, 
and the percentage of water absorbed was computed on the dry 


1921] GARDNER—GERMINATION 265 


weight basis. To confirm the results obtained a second series 
was treated similarly. Failing to obtain concordant results, two 
additional series of determinations were made. ‘The variation in 
time of the appearance of the first hypocotyls and the uneven 
surfaces of the seed coats account for much of the variation in the 
amount of water absorbed. The results are given in table X. 

In view of the small size of the seeds, their irregular surfaces, 
the difficulty of uniform drying, and the increase of weight on 
account of germination, the data of these determinations are not 


TABLE X 
SERIES I SERIES 2 SERIES 3 SERIES 4 
;Percent- Percent- Percent- . Percent-| . 
age of | No. of | age of | No. of | age of | No. of | age of | No. of 
SEEDS imbibed | sprouts | imbibed] sprouts | imbibed] sprouts | imbibed] sprouts 
water. water water water 
Light 
Nicotiana Tabacum...... Geugnl 730 | 62.3) ro, |ro8:5 Sn Os 25 8 
Verbascum Thapsus..... O44.) 122 «|-58-8 2M ie ot NO 2\23 6 
Weems Carota. ......... 114.5 2 92.4 4 S5e7 4 64.8 3 
Oenothera biennis....... 40.7 El. 43-5 A \ On, 7 Te Ou Als gilet 
PGmien CEISHIS.......... 48.4 2 Nees: 2 54-3 & ery, 29 
Darkness 
Nicotiana Tabacum...... 60.0 | 5.0 @ |967.7 I 63.6 ° 
Verbascum Thapsus..... BE s2 o | 68.0 o | 90.0 2 76.9 fo) 
Reames ArOta......... Pip GOs 7 S, 460s a a923 I 64.0 6 
Oenothera biennis....... AG @ se8.5 fb AG .4 Po | 4a.@ II 
PUIMESCHISPUS.......... Fave Sreigare Te 4on@ Lee hoe Neate) IO 


surprisingly discordant. In some cases the high percentages of 
water absorbed is accounted for by the many and large seedlings 
which could not be removed without more seriously changing the 
data. After eliminating the cases open to suspicion on account 
of the numerous sprouts, imperfect drying, etc., there appears to be 
relatively little difference in the percentage of moisture absorbed 
by seeds germinated in light and darkness. In Nicotiana Tabacum 
seeds of series 4 the imbibition is 65.5 per cent with 8 sprouts in 
light, while in series 3 the imbibition is 67.7 per cent with one 
sprout in darkness, from which it appears that germination may 
occur even though a smaller percentage of water is absorbed. A 


266 BOTANICAL GAZETTE [APRIL 


comparison of determinations of absorption by Verbascum Thapsus 
seeds in light (series 2) and in darkness (series 4) indicates the same 
general relations. The data for the other seeds show similar 
results. From this experiment it appears that light is not necessary 
for the absorption of sufficient water for germination. 


Injection of seeds with water 


De Vries (8), having abandoned variation of temperatures, 
high temperatures (40-50° C.), and other treatments of seeds of 
Oenothera biennis as means of securing complete germination, 
injected soaked seeds with water under pressure of 6-8 atmospheres, 
after which he frequently secured germination.of too per cent. The 


TABLE XI 
DARKNESS| THEN IN |DARKNESS| THEN IN 
Ss F FOR LIGHT FOR FOR LIGHT FOR UNTREATED 
SEEDS 17 DAYS 7 DAYS I5 DAYS | 7 DAYS 
a (0) (b) Light Darkness 
Verbascum Thapsus...... I 4 re) | 24 81 re) 
Daucuhs(Garotar:. .1u eee 6 Io 8 12 61 21 
Oenothera biennis........ 8 43 5 55 33 8 
RuimexCrispus)... 2 o.4),0o57 5 8 II 56 71 3 
Nicotiana, Fabacum >. . 2s. 4 54 5 60 83 ° 


seeds (table XI, a) accordingly were soaked overnight at a tempera- 
ture of 25-28° C., wrapped in filter paper, placed in water, exhausted 
of the air in their intercellular spaces by reducing the atmospheric 
pressure to 20 mm. for 1 hour, and then subjected to hydrogen 
gas pressure of 575-675 pounds per square inch for 24 hours. The 
seeds were then placed to germinate in darkness under the usual 
conditions. A second lot (0) was treated in the same way except 
that it was subjected to a pressure of 500-650 pounds per square 
inch for 48 hours. Both lots were germinated at room temperature. 
Evidently injection with water does not increase the germination of 
seeds of Oenothera biennis, Nicotiana Tabacum, Daucus Carota, or 
Rumex crispus in darkness. When the seeds are subsequently 
exposed to light, they germinate in one or both tests. These results 
confirm the conclusion arrived at in the weighing experiments, that 
impermeability to water is not the limiting factor in light germi- 


1921] GARDNER—GERMINATION 267 


nation. Perhaps better illumination of the injected Oe¢enothera 
biennis seeds made possible the increased germination reported 
by DE VRIEs. 
Increased oxygen supply 

In his investigation of the delayed germination of seeds of 
Xanthium, CROCKER (7) found that the seed coat excludes oxygen, 
while SHULL (46) found a very definite relation between the oxygen 
supply and the percentage of germination in seeds of Xanthium. 
In order to discover if increased oxygen supply would promote the 
germination of the light-sensitive seeds in darkness, the following 
experiment was performed. Counted seeds were placed on wet filter 
paper in open dishes and placed under water-sealed glass cylinders 
containing 40, 50, 60, 70, and 80 per cent oxygen respectively 
(table XII). Each cylinder was placed in a dark room at 23-28° C. 
and covered with a light-tight metallic cylinder. 


TABLE XII 
Percentage of germination in oxygen 
Seeds 
40 50 | 60 | 70 | 80 
Diicamana Pabactum........:.-..... ° aa ° O O 
Meteseemer Phapsus............... fe) ° ° fe) ° 
ce 3 3 9 15 24 
EMMENMPMMIA. 520 ..-...:..-.|2..-...- I I I 3 
DMEORISOI Se ie 19 18 23 


A comparison of the germination in darkness in the presence 
of different percentages of oxygen shows an increase of germination 
of seeds of Daucus Carota and Rumex crispus with an increase of 
oxygen supply. Other conditions in each of the cylinders being 
the same so far as known, this must be attributed to increased 
oxygen supply. A similar experiment with higher and lower per- 
centages of oxygen would have been interesting, especially a test 
of germination in 20 per cent oxygen (ordinary air) under these 
conditions. It would probably have given results similar to those 
in 40 per cent oxygen and would have been rather more conclusive. 
The regularity of the increased percentage of germination, however, 
due to increased concentration of oxygen, indicates the reliability 
of the results. Clearly this experiment does not indicate an oxygen 


268 BOTANICAL GAZETTE [APRIL 


deficiency in seeds of Nicotiana Tabacum, Verbascum Thapsus, and 
Oenothera biennts. 
Substrata 

LEHMANN (30) reported increased germination in darkness of 
light-sensitive seeds such as Ranunculus sceleratus with soil as 
substratum. Baar (1) obtained an increased percentage of 
germination of seeds of Amaranthus when he substituted earth for 
filter paper as a substratum, but OTTENWALDER (40), who used soil 
and sand as well as filter paper as substrata for his Epilobium 
hirsutum seeds, found beneficial results in his experiments with 
only one lot of sand. Investigation showed that the sand had 
been treated with acid which had not been thoroughly washed out. 


TABLE XIII 
PERCENTAGE OF GERMINATION AFTER 18 DAYS 


ON SOIL ON SAND In sort |IN SAND/ON FILTER PAPER 
SEEDS 
Light | Datk- | right | Datk- | right | Light | Light | Datk- 
Verbascum Thapsus oor 40 34 30 20 2 24 41 3 
Daucus Careta..2h. . 2.5% 56 28 54 28 32 58 | 63 19 
Oenothera biennis....... 2 fe) 2 2 fo) 6 * 2 
Rumiex crispus... 5.5.2.5 68 2 46 ike) 12 38 | 42 8 
Nicotiana Tabacum......| 70 6 78 ° 56 | 90° ee I 


In view of the divergent results with the different seeds, it was 
deemed desirable to determine the relation of sand and soil to 
germination in darkness of these light-sensitive seeds. Also the 
question arose as to whether light was as necessary under natural 
conditions as under laboratory conditions for the germination of 
light-sensitive seeds. The substrata were carefully sterilized, 
uniformly wetted, and prepared for the seeds. The seeds in 
‘“‘sand”’ and in ‘‘soil”’ were buried to a depth of 0.25 inch. All 
were put under the same temperature conditions (23-26° C.). The 
results are shown in table XIII. The experiment was begun 
May 13 and closed June 1. 

The germination of seeds of Verbascum Thapsus on soil and sand 
in darkness is somewhat higher than that on filter paper. The 
substratum appears to have exerted a slightly favorable effect on 
the germination of seeds of Daucus Carota, but none on the other 


1921] GARDNER—GERMINATION 269 


seeds. Such results suggest a beneficial effect on some particular 
constituent contained only in seeds of Verbascum Thapsus. In light 
the percentage of germination seems to correspond roughly to the 
light intensity. Where the lighting is good, as on the filter paper 
or sand, the germination is good. Where it is diminished, as in 
the case of seeds buried in sand or soil, the germination is reduced. 
The increase of germination of seeds on sand or soil in darkness 
may be referred largely to the action of constituents of the soil 
and sand. The low germination of seeds of Oenothera biennis 
may be due to periodicity in dormancy, since seeds from the same. 
lot gave a germination of 78 per cent in light and o per cent in 
darkness in October. From these results it appears that constitu- 
ents of soil may only partially substitute for light with some seeds 
and not at all with others. 


Effects of electrolytes 

The effects of electrolytes on the germination of light-sensitive 
seeds have been variously reported. Beneficial effects on germina- 
tion in darkness of Ranunculus sceleratus from hot water extracts 
of soils, Knop’s nutrient solution, and salt solutions have been 
reported by LEHMANN (30). He reported no benefit from cold 
water extracts of soils. LEHMANN and OTTENWALDER (36) found 
that weak acid solutions promote germination in darkness of 
seeds of Verbascum Thapsus, V. thapsiforme, and Lythrum Salicaria. 
OTTENWALDER (40) reported that acids promote the germination of 
seeds of Epilobium hirsutum in darkness. GASSNER (15) reported 
that nitrogen compounds such as nitrates, nitrites, and ammonium 
salts through a wide range of concentrations favor the germination 
of seeds of Chloris ciliata in darkness. As many of the electrolytes 
reported by these investigators belonged to the lyophobe or 
lyophile series, a systematic study of their effects was undertaken, 
to discover, if possible, some relation between electrolytes and germi- 
nation. Lots of too seeds each were counted into test tubes, about 
2 cc. of a solution of an electrolyte added, and the tubes placed in 
darkness. After 24 hours most of the solution was drained from 
each test tube, which was promptly returned to the dark chamber 
for the seeds to germinate. The period allowed for germination 
was 7 days after soaking. The results are given in table XIV. 


270 BOTANICAL GAZETTE [APRIL 
TABLE XIV 
GERMINATION IN DARKNESS IN VARIOUS CONCENTRATIONS 
z|\2|% aia? 
aE ge Z oe 
Ate Ne | or} ie At Se i ea 
4 re) hed 6) eS re 2 cs he 
SEEDS Z|. 0 ° o | o ° SEEDS a | 610 | owlter 
Acetic acid Sodium iodide 
Verbascum Thapsus....| o o| 77 | 79 | 86 Verbascum Thapsus...].... | ase 37° | OL Anee 
Rumex crispus......... oj), 0 |. 20.41 23 ° Rumex Crispus. 4). o-e lee eee (ee I 5 
Daucus Carota........ ° fo) I 3 4 Daucus Careta., . ..-+|/5. eee 4 3 ° : 
Oenothera biennis...... o./ 3] 48 j 424) 33 Oenothera biennis.....].... | ~ +e) 42) oe thee ; 
Nicotiana Tabacum....| o| o| 27 | 46] 23 Nicotiana Tabacum...].... Bee abe 2 3 ; 
Butyric acid Sulphuric acid 
Verbascum Thapsus....| o | o]| o | 80 | 86 Verbascum Thapsus...}....|.... 8 | 35 | 94 | 97 
Rumex crispus......... ° fo) ° 6 2 Rumexicrispus,..\.. -»-+1o. eee ° 3 ° ° 
Daucus Carota. =... ° ° ° I 6 Daucus Carota.......|....)-s7-)) aoe 3 7 
Oenothera biennis...... ° ° o | 45 | 50 Oenothera biennis.....]....|.... 25 | 36] 34 | 40 
Nicotiana Tabacum....| o ° o | 38 | Io Nicotiana Tabacum...|....|....]| 2 | 24 9 2 
Citric acid Potassium sulphate 
Verbascum Thapsus....|.... © | 40 | 95 | 08 Verbascum Thapsus...]....|.... 87 | So |100. | 4... 
Rumex'crispus.2..:.-.-|...- GM) Tz Srl as Rumex Cuspus.....5.-..-loo soto I ° I ; 
Daucus Carota....... o | Io ee lige 3 Daucus Carota . . . .; «|... eee ° I Ms 
Oenothera biennis......|.... 38 | 61 | 52 | 68 Oenothera biennis.....|....|.... 44 | 58 | 38 : 
Nicotiana Tabacum....}.... 36 | 35 | 54 | 58 Nicotiana Tabacum...]....].. 12 5 8 Z 
Tartaric acid Ammonium sulphate 
Verbascum Thapsus....|.... ©, | z4.| 8x 1753 Verbascum Thapsus...| © | 51*| 92 | 87 | 98 |.... 
Rumex/erispus....0...-<)...- 7 74 5C ° Rumex crispus........ ° 3 7 5 9 
Dauens Carotag eo ose. clei I rol We 8 Daucus Carota.......| 0 fo) I 4 | 15 : 
Oenothera biennis......|.... 2 | 52 | 757| 167 Oenothera biennis..... o | 44*)) S31 54 1eeaeieeee 
Nicotiana Tabacum....|....| 21 | 59 | 50 | 27 | Nicotiana Tabacum...} o | 36*] 44 | 40 | 23 Bs 
Malic acid Sodium sulphate 
| 
Verbascum Thapsus....|.... 5 | 64 |x00 | or | || Verbascum Thapsus...].... | oe oh oe | 94 | 88 3 
RANTES LCHISPUSs son.) sil ae A220) Teo, || Re: CxISPUS.= oc alee | «50h een 2 A 
Daucus Carotas ose scl. 2 5m Na: de (le Daneus Carota. . . .:..]..-feaes zr ° 1% 
Oenothera biennis...... 27 | 55 | 54 | 60 Oenothera biennis.....].... | «ee ares 48 | 41 5 
Nicotiana Tabacum....}. 40 | 43 | 31 | 83 Nicotiana Tabacum...]....|....] 4 9 | 10 ihe 
| | / i 
Potassium sulphocyanate Lithium sulphate 
Verbascum Thapsus....]....!.... | 80 | 75 | 88 .-|| Verbascum Thapsus...|]....|.... 03 | 91 | 90 2 
RiUMER CRISPHS. 2 0.2. 00] o 5 Pigs hg 2 2 RUMeEX EISPUS....  ...6 ofc os es te ° I 
Daweus Catota fe njse-rmleee = 8 4 2 Daucus Carota.......|....)..--| 2 3 ° : 
Ocnothera biennis.....<|.-=.|-.- 1) yom) Os 1 Gr | ! Oenothera biennis.....|.... WP ee oh ee 2 
Nicotiana: Fabacum.. 4)... <)> = 2¢ | 3 | I # | | Nicotiana Tabacum...]....|... | 17 | 12 9 : 


Verbascum Thapsus 
Rumex crispus..... 


Daucus; Carcta se ..< 


Oenothera biennis. . 


Sodium sulphocyanate 


| | i 

2 il Ce iesced Se inte Verbascum Thapsus 

PhS al p-Sa 1a | Rumex crispus..... 

eae a 4 O|.... Daucus Carota.. . .-..|-..=}--~» 
|. pon iGsot fey Saale cee Oenothera biennis. . 

Re Ot “Gt 744 Nicotiana Tabacdm. ..].....].- =< 


Nickel sulphate 


j 
sista oo ee | 6st 62 | 86 |.... 
fees Pees 3| 2] © oa 
5.| 28 | 22 |..-. 
oe” ee: tae 33. | 40 | BF dkenw 
2133 | O8ees 


* Little more than swelling. 


7 Injured. 


1921] GARDNER—GERMINATION 271 
TABLE XIV—Continued 
eS lie Z 
re A <a gre 
wie i.e t's 8 wot aot eS 
SEEDS Pato leit eh 6 SEEDS St) tan he. tiene) me 
Zinc sulphate Cobalt nitrate 
Verbascum Thapsus....}....].... 41 | 80 | 97 Verbascum Thapsus...]....|.... 31*| 99 | 93 
PRIME SICHISDUS 2 oc. nice o-oo e clls os ° ° fo) Rimex Crispuse: «,..-0..] aan lanes I 4 2 
PUN ANOtA 8... <|-...(|-52.| 2 I I Wanicus/Carotaist..1..c\lc1-aiee nol °4 2 8 
Oenothera biennis......|....|.... Bie Aon Sa Oenothera biennis.....|....|.... ASanS 7a eto 
Nicotiana Tabacum..,.|.... - 8 a 2 Nicotiana Vabacum).”..|..:.. |e!) £3) | 25) |e x0 
Potassium nitrate Potassium hydroxide 
Verbascum Thapsus....|....|.... 36 | 82 | o5 Verbascum Thapsus...].... o | 84 |100 | 98 
PSGIHEMIGEISDISE. 25... -|osas |e oa. 3 2 ° IRTHERCLISDUSi io a seine az ° 6 6 9 
ameus Carota.. 0.2 .6.]....)....| 8 | 78 8 Daucus: Carota .....5 sclace sj © 7 9 4 
Oenothera biennis......|....|.... Soe Or |) 55 Oenothera biennis.....].... ol 44 | 55 || 42 
Nicotiana Tabacum....]....|.... | 5o |) 23, | 23 Nicotiana Tabacum...|....| © | 17 | 34 | 53 
Ammonium nitrate Ammonium hydroxide 
| 
Verbascum Thapsus....| 0 | 62 |1co | 82 | 93 Verbascum Thapsus...}....].... T. (QO 405 
IRGUMexICHISDUS. 2.4. . <: Op -esi aa er 8 RGHeEXEHISPUS! Yes cele melee ate fo) ° I 
Daucus Carota........ ° ° fo) I I Watens Carotat. a Jondeeee les ao I 3 
Oenothera biennis...... ©; 70 | 66 | 68 | 64 Oenothera biennis.....]....|.... ane Seti SO 
Nicotiana Tabacum:...| 0o | 54 | 73 | 34 | 28 Nicotiana Tabacum...]....|.... Tee orage ler 
Sodium nitrate Sodium hydroxide 
| 
Verbascum Thapsus....| © | 64 |100 | 98 | or Verbascum Thapsus...].... ©) | 88, | G0) fos 
Rumex crispus......... o | 14 | 26 4 | 15 RUMER CLISPUSie<..c 2 cine ° I 2 2 
Wancus Carota........ ° 6 I 6 4 Danes Carotay.<. sos-\-4. 2) 10 ey | seis 6 
Oenothera biennis...... ©) || ‘Go! | 67:67" |).50 Oenothera biennis.....].... © | 50) |) 57 ° 
Nicotiana Tabacum....| o | 38 | 34 | 29] 11 Nicotiana Tabacum...|....| o | 21 Eh | ante, 
Aluminum nitrate . Hydrochloric acid 
Verbascum BIHAN SUS). <2 l| arene lei evel s 55 | 98 | 93 ..|| Verbascum Thapsus...].... Serceily + tok | eh | 61 
MMMERGEISOUS.......-. |. 2.0). o0- I I ° aOR UMeX: CLISDUS.< & 4.5). cls cn Secon LO | ° 
PeUeMeAtGtA......:|....)--..|° 2 3 9 ell OAICNs GarOtay.eiss 5 cloren ie oO 5 4 
Oenothera | SHCSVOWa Se ee eee te es T2) | $7.1 55 ..|| Oenothera biennis.....].... Sei ese a Oiren tan ) 
Macatiana Tabacum....|....|:... oro | 74. P | INicotianamlabachme sel.cosiiesss 1O>|isto) | 22 


* Little more than swelling. 


From table XIV it appears that organic-acids, bases, and salts 
of monovalent, bivalent, and trivalent ions induce germination in 
darkness of seeds of Verbascum Thapsus (80-100 per cent), Oenothera 
biennis (40-60 per cent), and Nicotiana Tabacum (10-50 per cent), 
while they inhibit the germination of seeds of Rumex crispus and 
Daucus Carota. These results were confirmed in an attempt to 
determine the minimum effective concentration of the electrolytes. 
In this attempt it was found that as good germination in darkness 


o.ocooor N 


272 BOTANICAL GAZETTE [APRIL 


occurs in ten-millionth normal solutions as in one-thousandth normal 
solutions. ‘These results indicate no definite relation between the 
nature of the ion and germination. In another series of experiments 
on the relation of electrolytes to germination, with seeds from 
another crop, it was found that the germination of Verbascum 
Thapsus, Oenothera biennis, and Nicotiana Tabacum was inhibited, 
while the germination of Rumex crispus seeds was promoted in 
darkness by the action of the various electrolytes. This suggests 
that something in the conditions of growth, maturing, harvesting, 
or storage may have changed the sign of the charge of the ionizable 
constituents of the seeds. Further work on the effects of electro- 
lytes on the germination of these seeds is highly desirable. 


Soaking in solutions of electrolytes 


It is generally believed that forcing agents of germination such 
as light, enzymes, and electrolytes are most effective during the 
early stages of incubation. KiInzEL (24) by soaking seeds of 
Nigella sativa in a solution of papayotin and asparagin for 5 hours 
and then in water for 24 hours secured a 30 per cent increase of 
germination of “light hard” seed. OTTENWALDER (40) has reported 
that 24 hours is not sufficient time to secure the full effect of the 
acid on the germination of seed of Epilobium hirsutum, and that 
about 48 hours’ soaking was necessary to get the best results from 
the action of the acid. An attempt was made, therefore, to 
determine whether soaking in solutions of electrolytes could pro- 
mote the germination of light-sensitive seeds. 

Seeds were soaked in the various solutions for 24-28 hours and 
washed in distilled water until all of the solution was removed. 
To avoid light effects, care was taken to work in very diffuse light. 
The seeds were spread on filter paper in Petri dishes and placed to 
germinate in light and darkness respectively, at room temperature, 
for 8 days. 

An examination of table XV A shows that soaking in rather strong 
solutions of hydrochloric acid promotes the germination in dark- 
ness of seeds of Nicotiana Tabacum, Verbascum Thapsus, Oenothera 
biennis, and Rumex crispus, while it hinders the germination of 
seeds of Daucus Carota in light. ‘The beneficial effects of solutions 


1921] GARDNER—GERMINATION 273 


of sulphuric acid appear only in the germination of seeds of Daucus 
Carota in darkness. Soaking seeds of Nicotiana Tabacum and 


TABLE XVA 
LIGHT DARKNESS 
| 
SEEDS N o.1N 0.01 ote) N o.r N ie-eeny | 0 


Hydrochloric acid (soaked 26 hours) 


Nicotiana Tabacum 
Verbascum Thapsus 
Manpens Carota..........: 
Oenothera biennis....... 
Memes CrISDUS........4: 


Macedana Tabacum......!....: 

Werpascum Thapsus: ....|...:.. 
1 TE GA o_o 
Oenothera biennis.......|...... 
eeReNCrISIS =... ....2)-..--- 


Nicotiana Tabacum...... 
Verbascum Thapsus..... 
Meee Catoia........:... 
Oenothera biennis....... 
Pemex eriSpUS........ .*. 


Sodium hydroxide (soaked 24 hours) 


Nicotiana Tabacum......}| 0 81 61 74 ° 26 25 IQ 
Verbascum Thapsus..... ° 63 86 83 fo) ime) 19 5 
Mies Carota.......... ° 77 63 75 ° 30 45 26 
Oenothera biennis..... Patines : 87 42 51 ° 22 ‘2 12 
umex eCrispuS.......:.. ° 59 76 |) 68 ° 17 26 24 


TABLE XVB 


PERCENTAGE OF HYDROGEN PEROXIDE (SOAKED 27 HOURS) 


SEEDS Light Darkness 
50 20 Io 5 co | 50 20 Io 5 foye) 
NMicotiaea Vabacum.......-...... Beh Zeb 7a pS, fad (On b seul St. |. 46 '| to 
Weenasemm Thapsus...:........ Tate7o.b ve 1 680 fio G4 0 |). 36 2 5 
LS TO OS |..04.) ge 9s p20 Pawel OR i 5201 26 
Oenothera biennis............... £6 ad sa ae pCa en Re x > Nop fae 


Sete CISPUG.. .. 2... 2. 25.2-.. 731 66,1 73 77.) Ga PAs SO 77a: son) 24. 


274 BOTANICAL GAZETTE [APRIL 


Rumex crispus in solutions of sodium sulphocyanate appeared to 
promote their germination in darkness. Soaking in potassium 
sulphocyanate gave similar results. Soaking in solutions of hydro- 
gen peroxide promoted the germination of seeds of Nicotiana 
Tabacum, Daucus Carota, and Rumex crispus in darkness (table 
XV 8B). Germination in o.co1 N sodium hydroxide was about the 
same asino.orN. Soaking in solutions of sodium hydroxide gave 
increased germination in darkness of seeds of Verbascum Thapsus, 
Daucus Carota, and Oenothera biennis. ‘These results were con- 
firmed by another set of tests. 

The reaction of the seeds to the different electrolytes indicates 
that the ions of the electrolytes are acting on different constituents 
of different seeds. While, as shown in the preceding experiment, 
the use of hydrochloric acid, sodium sulphocyanate, and hydrogen 
peroxide as substrata yields no increase of germination of Rumex 
crispus in darkness, this experiment shows that soaking for a short 
period (24-28 hours) in solutions of these electrolytes does pro- 
mote their germination in darkness. As Rumex crispus seeds were 
brought to germination in darkness by abrading and removing the 
coats, and by the action of concentrated sulphuric acid, their 
germination may naturally be referred to coat effects, the com- 
pounds acting on some constituent of the coat. The germination 
of Daucus Carota is not so easily accounted for. The germination 
in darkness was only slightly promoted by increased oxygen supply 
and on soil as substratum. The hydrogen peroxide may yield an 
increased oxygen supply and thus promote the germination of these 
seeds, but an explanation of the beneficial effects of sulphuric acid 
and sodium hydroxide on the same material is not easily made 
unless we refer to coat effects which have not been clearly indicated 
by other treatment. A longer period of soaking (48 hours) might 
have yielded data to settle this, as well as the failure of germination 
of the other seeds in certain solutions. 


Lipoid solvents 


Finding that lipoids occur in the coats and embryos of all the 
seeds and in the endosperm of four of them, it was thought desirable 
to determine the effect of acetone, alcohol, and ether on their 


1921] GARDNER—GERMINATION 275 


germination. Seeds of each kind were soaked in acetone for 15, 30, 
and 60 minutes respectively, air dried for two hours, and placed 
under favorable conditions for germination in darkness. At the 
end of 8 days of incubation (in darkness) the seeds were placed in 
light for 8 days, where none of them germinated. Other seeds 
were similarly treated with alcohol with similar results. A few 
seeds of Daucus Carota and Oenothera biennis survived the alcohol 
treatment and germinated in light and darkness. Other seeds were 
treated with ether, as indicated in table XVI. 

The results show no promotion of germination of light-sensitive 
seeds in darkness when treated with lipoid solvents, but rather show 
inhibition or diminution of subsequent germination in light. This 
is especially true for acetone and alcohol. Ether inhibited germina- 


TABLE XVI 
SOAKED IN ETHER SOAKED IN ETHER SOAKED IN ETHER 
SenpS I5 MINUTES 30 MINUTES 60 MINUTES 

Darkness Light Darkness Light Darkness Light 
Verbascum Thapsus...... fe) 28 ° 30 fe) ° 
MeStMeX CHISPUS........... ° 35 ° 3 ° 2 
Wancus Carota... 0s... .-. 15 17 17 iy 12 12 
Oenothera biennis........ ° 76 ° 74 ° 62 
Nicotiana Tabacum....... ° 43 fe) 64 ° eal 


tion in darkness of all seeds except Daucus Carota, and diminished 
the subsequent germination in light of seeds of Verbascum Thapsus, 
Rumex crispus, and Nicotiana Tabacum. Ether treatment did not 
affect the subsequent germination of seeds of Oenothera biennis in 
light. : 
Microchemistry 

In an attempt to find the substance responding to the action 
of light, an examination of the seeds was undertaken by micro- 
chemical methods suggested by ECKERSON (g), and the nature and 
distribution of the different structural and nutritive materials were 
determined. Much of the information thus obtained has no evi- 
dent bearing on the problem of light germination and may best be 
presented in a separate publication. Some of the substances and 
conditions in these seeds which may function in light germination 
are fat, suberin, starch, and reaction. 


276 BOTANICAL GAZETTE [APRIL 


Starch occurs in the endosperm of seeds of Rumex crispus and 
Daucus Carota. It does not occur in the embryo of any seeds under 
investigation. Moreover, the hydrogen ion concentration is not 
likely to be materially changed by such hydrolysis of starch as 
may occur during their germination, and therefore we need not 
consider starch an important factor in their germination. Since 
suberin is found in the coats of Oenothera biennis and Daucus 
Carota, but not elsewhere, it can hardly be considered a common 
limiting factor in the germination of the light-sensitive seeds 
studied. Oily or fatty substances were found in the cell contents 
of coats, endosperms, and embryos of each of the seeds, appearing 
as small.droplets of substance readily stained with Soudan III or 
Scharlach R. These lipoids almost never occurred as continuous 
layers which might obstruct the entrance of water or other substance 
necessary for germination, but usually as emulsions of fats in the 
cellsap. The results of the experiments on the absorption of water 
support the observation that there is no important obstruction of 
water by the constituents of the coats. Hence suberin and lipoids 
need no further consideration as limiting factors in absorption of 
water by these seeds. 

Using neutral red as an indicator, seeds soaked in water in 
light and in darkness, as well as dry seeds, were tested for the 
reaction of the different parts with results shown in table XVII. 

The outstanding result of the microchemical examination. is 
the greater acidity of seeds incubated in light as compared with 
those incubated in darkness. This was found to be the case in each 
of the five kinds of light-sensitive seeds. ‘The embryos incubated 
in light had a higher hydrogen ion concentration than those of the 
same kind incubated in darkness. ‘This was especially noticeable 
‘in the hypocotyls. This result is contrary to HEINRICHER’S (21) 
unsupported assumpton that the effectiveness of the fat splitting 
lipase was favored by the increased acid formation in darkness in 
Phacelia tanacetifolia. Moreover, HEINRICHER offered no experi- 
mental evidence of increased acid formation in darkness. Having all 
known external factors, except illumination, alike for the seeds under 
investigation, we may properly conclude that the varying factor, 
light, in some way brings about increased acidity of their embryos. 


1921] 


SEEDS Coats 
Ti ares Acid 
Soaked in dark- 

SNR gp Wale xis Acid . 
Soaked in light. .}| Acid 
iy ne Acid 
Soaked in dark- 

MESS os k's wk Acid 
Soaked in light. .| Acid 


Outer walls 
acid; inner 
alkaline 

Soaked in dark- 


ESE clea s Outer acid; 
inner le 
line 

Soaked in light. .| Outer acid; 
inner alkaline 
LD eer Acid 
Soaked in dark- 
MESS. MH. .... 5 Acid 
Soaked in light. .}| Acid 


i 


GARDNER—GERMINATION 


TABLE XVII 


ENDOSPERM 


Verbascum Thapsus 


Cell walls acid; con- 
tents neutral 

Cell walls acid; con- 
tents neutral 

Walls acid; . contents 


acid 
Rumex crispus 


Outer layer Wie ere et oe edt ical | Walle aid contents new. cell 


contents neutral 


Outer layer acid; con- 
tents mostly acid 


Walls acid; 
acid 


contents 


Daucus “OSE T, AG Fey a BOR Ee a ar ne 


Cell walls acid to alka- 
line; contents neu- 
ir al to alkaline 


Walls acid to alkaline; 
contents neutral to 
alkaline 


Walls acid to alkaline; 
contents neutral 


Nicotiana a a eee ee 


Cell walls acid; con- 
tents neutral to alka- 
line 


Walls acid; contents 


alkaline to neutral 


Walls acid; contents 
neutral to acid 


EMBRYO 


Cell walls acid; 
tents neutral 


con- 


Outer cell walls and con- 
tents alkaline to neu- 


tral; inner cell walls 
acid: contents alka- 
line 


Walls and contents acid 


Walls and contents neu- 
tral 


Walls acid; 


neutral 


contents 


Walls and contents acid 


Alkaline 


Alkaline except at tip of 
hypocotyl 


Walls and contents acid 


Cell walls acid; con- 
tents neutral to aika- 
line 


Cell walls acid; con- 
tents partly alkaline, 
partly neutral 


Walls acid; contents 
acid except at base of 
cotyledons 


278 BOTANICAL GAZETTE [APRIL 
TABLE XVII—Continued 


SEEDS Coats ENDOSPERM EMBRYO 


Oenothera biennis 


Diy. alae Partly acid Slightly acid or neutral | Cell walls acid; con- 
tents neutral 


NESS Yn wes bee Mostly acid Slightly acid to neutral | Cell walls acid to neu- 


tral; contents alkaline 
to neutral 


Soaked in light. .| Partly acid Neutral to acid Walls and contents acid 


Quantitative determination of acidity 


To verify the results of the microchemical examination, 5 gm. 
of each kind of seed incubated in light for five days and 5 gm. of 
each kind incubated in darkness for five days were separately 
ground, digested in neutral alcohol and ether, and then titrated 
with N/1o NaOH. The results obtained were as follows: 


Seed Light Darkness 
PRUMMIEK CEPSUSY bo opens ste ee 4.5 ec. 2.8 ce 
Daucus Cate 5 oes reer as ces 2.8 
Verbascuin Thapsus. ... 2. o..% +. 4.8 4.4 
Nicotiana Tabacuim... 0 5 008 4. 3.9 2:6 


These results show greater acidity of seeds incubated in light 
than of those incubated in darkness, and confirm the findings of 
the microchemical examination. While the increase of titratable 
acidity was not large, it was measurable and repeatedly obtained, 
and apparently was sufficient in each instance to determine germina- 
tion. Since light is the variable factor in this and the preceding 
experiment, we may properly conclude that light initiates changes 
which produce the increased acidity of seeds incubated in light 
over those incubated in darkness. These results establish the fact 
that light functions in some way to bring about increased acidity in 
these light-sensitive seeds. There remains to show in the following 
experiment, if possible, how the acidity is increased. 


Effect of germination on substratum 


Having found that the embryos of these seeds become acid in 
reaction by incubation in light, it was thought that testing the 


ee 


1921] GARDN ER—GERMINATION 279 


reaction of the substratum after a period of incubation might 
throw some light on what was happening in the seeds. A small 
quantity of each kind of seed was soaked in distilled water, 
o.o1 N NH,NO, solution, and o.or N NaNO, solution at room 
temperature, and the substratum tested for reaction with neutral 
red. The results are shown in table XVIII. 


TABLE XVIII 


SOAKED 18 HOURS IN | SOAKED 24 HOURS IN | SOAKED 24 HOURS IN 


DISTILLED WATER o.o1 N NH;NO; o.orN NaNO, 
SEEDS i  ( 
Light Darkness Light | Darkness Light Darkness 
Verbascum Thapsus...... Alkaline |Alkaline |Alkaline |Alkaline | Alkaline |Alkaline 
Eigse erispus........... Acid Acid Acid Acid Acid Acid 


| Alkaline |Alkaline |Alkaline Alkaline | Alkaline |Alkaline 
Neutral |Neutral |Alkaline |Neutral |Alkaline |Neutral 
Oe ANG Eee ip Waker etl it's be Alkaline |Alkaline 


Magtes Carota........- 
Oenothera biennis........ 
Nicotiana Tabacum....... 


From the results it appears that seeds of Verbascum Thapsus, 
Daucus Carota, and Nicotiana Tabacum excrete an alkaline sub- 
stance in darkness as well as in light; that seeds of Rumex crispus 
excrete an acid substance in darkness as well as in light; and that 
seeds of Oenothera biennis excrete an alkaline substance in light. 

A quantitative experiment also verifies a part of these results. 
Weighed quantities of each of the five kinds of seeds were incubated 
in light for five days in tubes containing 2 cc. of o.or N NaNO, 
respectively. The results are given in table XIX. 


TABLE XIX 


Seeds Weight of seeds (gm.) Required to titrate 


a... | 

Wereascom Phapsus.:............ °. 7406 t.o cc. o.or N HCl 
Pee BMSBN6 2). 5. . 2... ee ss 1.3385 1.5 cc.o.or N NaOH 
Lol 2D / 0.6510 0.9 cc. o.or N HCl 
Oeaetter pienmis,.............:. 0.7580 G.5ce, 0.007 N.HCl 
Titeomana fabacum.,............. | ° £6 £650.08 Ni EICl 


. 8403 


The longer period of incubation evidently allowed time for 
the excretion of a measurable amount of acid or base by each kind 
of seed. From these results it appears that seeds of Rumex crispus 
excrete measurable amounts of acid substance during a 
while the other kinds excrete alkaline substances. 


280 BOTANICAL GAZETTE [APRIL 


Enzymes 


The favorable effects of light on the germination of seeds of 
Veronica peregrina in the early work of HEINRICHER (19) was 
referred to its effect upon chemical actions connected with the 
reactivation of reserve materials, and later (21) to its effect upon 
enzyme activity in the production and digestion of stored foods. 
He (22) referred the retarding effect of light on the germination of 
seeds of Phacelia tanacetifolia to its photochemical action on 
reserve materials, and assumed that the effectiveness of the fat 
splitting lipase was favored by the increased acid formation in 
darkness, while the irrefrangible light or the rays of the first half 
of the spectrum interfered, neutralizing the acid and thereby 
checking the decomposition of fat. 

It seemed possible that enzymes of some kind might be active 
agents, and light the stimulus or trigger in the germination of 
certain seeds. Just what kinds of enzymes function most in the 
germination of light-sensitive seeds has not been shown. To 
determine whether proteolytic enzymes were the important enzymes 
for the seeds, as LEHMANN and OTTENWALDER (36) believed for 
seed of Epilobium lirsutum, seeds of Verbascum Thapsus and 
Nicotiana Tabacum were incubated in light and darkness 
respectively for four days and promptly. ground in a little 50 per 
cent water solution of glycerine to which a crystal of thymol had 
been added. Small drops of extract from each kind of seed were 
put on nutrient gelatin according to the method of GIESEN (16). 
After 30 minutes the extract was taken up with soft filter 
paper. ‘There were very shallow pits formed where the extracts 
incubated in light had been. There were also shallow pits formed 
on the gelatin where the extract incubated in darkness had been. 
In fact, the pits produced by the extract germinated in darkness 
were deeper than those produced by the extract incubated in light. 
To verify these observations, the tests were repeated after allowing 
a more complete extraction of the enzyme. ‘The extracts of seeds 
incubated in light were put in light, and the extracts of seeds 
incubated in darkness were put in darkness. The following day 
the same tests were repeated. Extracts from each lot of seeds 
were tested for their action by putting loopfuls on gelatin. After 
30 minutes the extracts were removed separately, when it was 


192T] GARDNER—GERMINATION 281 


found that well defined pits had been formed in the gelatin. More- 
over, where the extract was left on the gelatin for 24 hours, 
the pits became quite deep, even though there was abundance of 
thymol to inhibit bacterial action. These results were confirmed 
by the method of Griiss (17). 

Since the activity on gelatin of enzymes of seeds incubated 
in darkness was equal to or greater than that of seeds incubated 
in light, the favorable effect of hight on germination of Nicotiana 
Tabacum and Verbascum Thapsus cannot be referred to activation 
of proteolytic enzymes. ; 

It has already been seen that starch does not occur in the 
embryos of any of these light-sensitive seeds, and that it occurs 
only in the endosperms of Daucus Carota and Rumex crispus. 
From these facts it is evident that hydrolysis of contained starch 
can increase the hydrogen ion of the embryos little if any. It has 
been seen that proteolytic enzymes develop equally well in darkness 
and light in these seeds, hence they can be rejected as important 
factors in determining light germination. Also, incubation in 
light does result in increased acidity of embryos over those incu- 
bated in darkness. | 

It has been shown that the embryos of these seeds all contain 
fatty substances. ‘The generally accepted method of demonstrating 
the presence of lipolytic enzymes is by the increase of acidity in 
the presence of fats. Inasmuch as development of acidity in the 
presence of light and fatty substance has been clearly demonstrated, 
it may be concluded that light activates the lipolytic enzyme to 
split the fatty substance to yield an acid. The results obtained 
with enzymes of seeds of Verbascum Thapsus and Nicotiana Tabacum 
do not support HEINRICHER’S (22) assumption that light inhibited 
the action of lipase in seeds of Phacelia tanacetifolia. On the 
other hand, the results indicate that light favored the action of 
lipase in seeds of Verbascum Thapsus and Nicotiana Tabacum. 


Discussion 
CoAT EFFECTS 


The light relation of seeds of Rumex crispus is largely one 
affecting the coats, as is indicated by increased germination in 
darkness following abrasion and removal of coats, treatment with 


282 BOTANICAL GAZETTE [APRIL 


concentrated sulphuric acid, and increased oxygen pressure. 
Light may bring about some change in the coats of Rumex crispus 
to admit oxygen or other required substance, or permit the escape 
of some inhibiting substance such as an organic acid. It may 
change the relation of the lipoids from the oil water phase to the 
water oil phase, or break up a nearly continuous oil layer in the 
coat, thus allowing entrance or escape of some limiting factor. 
The presence of lipoids in the coats and the excretion of an acid 
instead of an alkaline substance during germination suggest that 
an enzyme acting in the coats hydrolyzes the lipoids, thus yielding 
acid and making the coats permeable to some required substance, 
or permitting the elimination of some inhibitory substance. 

There is some evidence of a coat effect in the germination of 
seeds of Oenothera biennis. While abrasion of the coats does not 
yield increased germination, hot water treatment and sulphuric 
acid treatment both yield considerable increases of germination in 
darkness. The presence of lipoids in the coats suggests the same 
explanation of the action of light as in the seeds of Rumex crispus, 
with the addition that the light may also have a beneficial effect 
on the constituents of the embryo. 

In the seeds of Nicotsana Tabacum, Verbascum Thapsus, and 
Daucus Carota there is little evidence of coat effects, there being 
no increased germination caused by abrasion, -sulphuric acid 
treatment, hot water treatment, or increased oxygen pressure. 
The only results suggesting coat effects are increased germination 
of Daucus Carota and Nicotiana Tabacum when soaked in hydrogen 
peroxide. This increased germination might be referred to the 
effects on the embryos. 

The seeds of this investigation fall into three groups. ‘The first 
is represented by the seeds of Rumex crispus, in which the coats 
must be made permeable to some external or internal substance 
by light, abrasion, or other agency before abundant germination 
occurs. The second group is represented by the seeds of Oenothera 
biennis, whose germination is partly dependent on the coats being 
made permeable, and partly on the activation of the embryos by 
light or chemical agencies. The third group is represented by seeds 
of Nicotiana Tabacum, Daucus Carota, and Verbascum Thapsus, 


1921] GARDNER—GERMINATION 283 


whose germination is not increased simply by making the coats 
permeable, but requires the action of light or a suitable substitute 
to induce good germination. 


MICROCHEMISTRY 


The results of the various mechanical, physical, and chemical 
treatments of the light-sensitive seeds have offered few suggestions 
as to the nature of the effects of light on their constituents in 
inducing germination. ‘The substitution of these various agencies 
for light has contributed little to an acceptable explanation of how 
light functions to bring about germination. These treatments, 
however, have served to localize the action of light and to determine 
the part of the seed affected. On the other hand, the microchemical 
examination yielded results which point to an acceptable explana- 
tion of the action of light on light-sensitive seeds. ‘The outstanding 
findings of the microchemical studies were abundance of lipoids in 
each kind of seed and increased acidity of seeds incubated in light. 
Thus there are linked together light, lipoids, and increased acidity. 


ENZYMES 


Since starch and other carbohydrates were not found in the 
embryos of these seeds and in the endosperms of but two of them, 
it is not necessary to give serious consideration to the probable 
reaction of the products of their hydrolysis. Moreover, since 
proteolytic enzymes were found to be equally active in light and 
darkness in Nicottana Tabacum and Verbascum Thapsus seeds, 
they need not be considered as important causes of increased 
acidity of the seeds incubated in light. It remains to be considered 
whether the products of the hydrolysis of the lipoids are the cause 
of the increased acidity in light. 

The development of acidity in the watery extract of an oily 
seed like that of the castor bean is generally considered evidence 
of the presence of lipase. Such development of increased acidity 
in light was demonstrated quantitatively for four of the five 
kinds of seeds, thus confirming the results of the microchemical 
‘examination, and giving reasonable ground for inferring that lipase 
splits the fats thus yielding fatty acids in seeds germinated in light. 


284 BOTANICAL GAZETTE [APRIL 


Again, the presence of neutral or very faintly alkaline fats in 
the cells of the dry embryos and the development of acids in 
embryos incubated in light and no change or development of slight 
alkalinity when incubated in darkness is very significant. It is 
generally assumed that increased acidity of fatty substances indi- 
cates hydrolysis due to the action of enzymes. If this assumption 
be granted, the admission is necessary that light initiates processes 
which in some way result in increased acidity, which is followed by 
germination, and that where light is not admitted acidity does not 
develop sufficiently to cause good germination. ‘Three explanations 
of how light acts may be offered: (1) light may act directly to 
split the fats to fatty acids and glycerine; (2) light may activate 
the lipolytic enzyme which splits the fats; (3) light may initiate 
some change that produces a little acid which may activate the 
lipolytic enzyme which splits the fats. 

While it is possible and even probable that light can act directly 
on the inclusions of cells to produce such changes as the formation 
of acid, it is hardly necessary to make this assumption. The 
proteolytic enzymes become active in the absence of light when the 
seeds are put under the usual conditions for germination. A certain 
percentage germinate in darkness even though they have not been 
treated with electrolytes or other stimulating agencies. Moreover, 
the amount of acid produced, though small; is relatively more than 
might be expected from the chemical action of light on lipoids. 
The evidence of the existence of lipase rather indicates either of the 
other explanations as much more acceptable. There is little choice 
between light activating the enzyme and light initiating some 
change which produces a little acid which may activate the lipolytic 
enzyme which splits fats. In either case light is the stimulus 
which initiates the changes leading to germination. 


Summary 
t. The seeds of Rumex crispus, Datura Stramonium, and 
Phoradendron flavescens were found to be light sensitive. The 
germination of seeds of Rumex crispus and Phoradendron flavescens 
was promoted by light; the germination of seeds of Datura Stra- 
monium was hindered by light. 


1921] GARDNER—GERMINATION 285 


2. Abrasion and removal of coats (ovary walls) of Rumex crispus 
seeds promoted their germination in darkness. 

3. Treatment of seeds of Rumex crispus and Oenothera biennis 
with concentrated sulphuric acid caused an increase in the per- 
centage of germination in darkness. 

4. Noreciprocal relation between the effects of light and temper- 
ature was found. 

5. Light was not necessary for the absorption of sufficient 
water for germination. 

6. Injection of water did not yield increased germination in 
darkness. | 

7. Almost all kinds of single electrolytes, regardless of the nature 
of the ions, seemed to promote germination of seeds of Oenothera 
biennis, Nicotiana Tabacum, and Verbascum Thapsus in darkness. 

8. Embryos of seeds incubated in light became more acid than 
those incubated in darkness. 

9. Light seemed to activate lipolytic enzymes which hydrolyzed 
fats to fatty acids. 

to. The germination of seeds of Ruwmex crispus in darkness was 
promoted (increased) by hot water treatment, abrasion, treatment 
with concentrated sulphuric acid, increased oxygen pressure, 
fluctuating temperatures, and soaking in solutions of hydrochloric 
acid, sodium sulphocyanate, and hydrogen peroxide. 

11. The germination of seeds of Nicotzana Tabacum in darkness 
was promoted by soaking in solutions of hydrochloric acid, sodium 
sulphocyanate, and hydrogen peroxide, as well as by the use of many 
single electrolytes as substrata. 

12. The germination of seeds of Verbascum Thapsus in darkness 
was promoted by the action of light, fluctuation of temperature 
during incubation, alternating high and low temperatures, soil, and 
many single electrolytes as substrata. 

13. The germination of seeds of Oenothera biennis in darkness 
was promoted during certain seasons by hot water treatment, 
sulphuric acid, preliminary incubation at low temperature, incuba- 
tion in alternating high and low temperatures, and single electro- 
lytes as substrata. 


286 BOTANICAL GAZETTE [APRIL 


14. The germination of seeds of Daucus Carota in darkness was 
promoted by increased oxygen pressure and preliminary incubation 
at low temperature, while it was hindered by soaking in hydro- 
chloric acid and by the use of single electrolytes as substrata. 


The writer gratefully acknowledges the kindly advice and direc- 
tion of Professor F. C. NEwWcoMBE, in whose laboratory this 
investigation was begun; and also the valuable suggestions and 
criticisms of Professor WILLIAM CROCKER, under whose direction 
this investigation was completed. 


ALABAMA POLYTECHNIC INSTITUTE 
AUBURN, ALA. 


LITERATURE CITED 


1. BAAR, H., Uber den Einfluss des Lichtes auf die Samenkeimung und seine 
Abhiangigkeit von andern Factoren. Sitzungsber. Kaiserl. Akad. Wiss. 
Wien. Math:-Naturwiss. 121:667—705. 1912. 

2. Becker, H., Uber die Keimung verschiedenartiger Friichte und Samen 
bei derselben Spezies. Beih. Bot. Centralbl. 1912:1-129. 

3. Bessey, E. A., The Florida strangling figs. Rep. Mo. Bot. Garden 
19: 25-33. IQ00. ! 

4. Caspary, M., Bulliarda aquatica DC. Schr. Physik. Okonom. Gesells. 
Konigsberg. 1866. pp. 66-82. 

, Uber Samen und Keimung von Pinguicula vulgaris. Schr. Physik. 
Gkonon. Gesells. Kénigsberg. 1867. p. 17. 

6. CIESLAR,» ADOLF, Untersuchung iiber den Einfluss des Lichtes auf die 
Keimung der Samen. Forsuch. Geb. Agrik. Physik. 6:270-295. 1883. 

7. CROCKER, WM., Role of seed coats in delaying germination. Bor. Gaz. 
AZ 1205-201. 1000: 

8. DE Vries, Huco, Uber kiinstliche Beschleunigung der Wasseraufnahme 
in Samen durch Druck. Biol. Centralbl. 35:161-176. rots. 

9. EckEersoN, SopHIA H., A physiological and chemical study of after- 
ripening. Bot. GAZ. 55: 286-299. 1913. 

10. Ficpor, W., Uber den Einfluss des Lichtes auf die Keimung der Samen 

elniger Gesnetiacsen Ber. Deutsch. Bot. Gesells. 25: 582-585. 1907. 
, Weitere Untersuchungen iiber den Einfluss des Lichtes auf die 
Keimung der Gesneriaceen. Ber. Deutsch. Bot. Gesells. 30:648-653. 1912. 
12. GASSNER, G., Uber Keimungbedingungen einiger siidamerikanischer 
Gramineensamen. Ber. Deutsch. Bot. Gesells. 28:350. 1910. 

, Untersuchungen tiber die Wirkung des Lichtes und des Tempera- 

turwechsels auf die Keimung von Chloris ciliata. Jahrb. Hamberg. Wiss. 

29: IQII. 


Il. 


12; 


1921] GARDNER—GERMINATION 287 


14. 


I5. 


32. 


GASSNER, G., Beitriige zur Frage der Lichtkeimung. Zeitschr. Bot. '7:609- 
661. 1015. 

, Einige neue Faille von Keimungsauslésender Wirkung der Stick- 
stofiverbindungen auf lichtempfindliche Samen. Ber. Deutsch. Bot. 
Gesells. 33: 217-232. IQIS5. 


. Gresen, R., Uber die proteolytischen Fermente der als Futtermittel 


benutzten Kornerfriichte. Inaug. Diss. Univ. Bern. pp. 34. 1909. 


. Griiss, J., Biologie und Kapillaranalyse der Enzyme. 1912. 
. Haack, —, Die Priifung des Kiefernsamens. Zeitsch. Forst- und Jagd- 


wesen. pp. I-64. I9QI2. 


. HernrIcHER, E., Ein Fall beschleunigender Wirkung des Lichtes auf die 


Samenkeimung. Ber. Deutsch. Bot. Gesells. 17: 308-311. 1890. 

, Notwendigkeit des Lichtes und befordernde Wirkung desselben 
bei der Samenkeimung. Beih. Bot. Centralbl. 13:164-172. 1902. 

, Die Beeinflussung der Samenkeimung durch das Licht. Wiesner 
Festschrift. Wien. 263-279. 1908. 

, Keimung von Phacelia tanacetifolia Benth. und das Licht. Bot. 
Zeitschr. 67:47-66. 1909. 


. Jonsson, B., Jaktagelsen of ver Ljusetse Betydelse for Frens Groning. 


Lunds Univ. Arsskr. 29:40-47. 1893. 


. Kryzet, W., Uber den Einfluss des Lichtes auf die Keimung. Ber. 


Deutsch. Bot. Gesells. 25: 269-276. 1907. 

, Lichtkeimung: Ejinige bestatigende und erganzende Bemerkungen 
zu den verlaufigen Mittheilungen von 1907-1908. Ber. Deutsch. Bot. 
Gesells. 261:631-645. 1908. 

, Lichtkeimung: Weitere bestatigende und erginzende Bemerk- 
ungen zu den vorlaufigen Mittheilungen von 1907-1908. Ber. Deutsch. 
Bot. Gesells. 261:655-665. 1908. 

, Lichtkeimung: Erlauterungen und Erganzungen. Ber. Deutsch. 
Bot. Gesells. 2'7:536-545. 19009. 


. LASCHKE, W., Einige vergleichende Untersuchungen iiber den Einfluss 


des Keimbettes sowie des Lichtes auf die Keimung verschiedener Samereien. 
65: 295-300. 1907. 


. LAURENT, E., Sur le pouvoir germanatif des graines a la lumiére solaire. 


Compt. Rend. Acad. Sci. 29:1295-1298. 1902. 


. LEHMANN, E., Zur Keimungs-physiologie und Biologie von Ranunculus 


sceleratus und einiger anderen Samen. Ber. Deutsch. Bot. Gesells 27: 476- 
494. 1900. 

, Lemperatur und Temperaturwechsel in ihrer Wirkung auf die 
Keimung lichtempfindlicher Samen. Ber. Deutsch. Bot. Gesells. 29: 577- 
589. IQII. 

, Uber die Beeinflussung der Keimung lichtempfindlicher Samen 
durch die Temperatur. Zeits. fiir Bot. 4:465-529. 1012. 


288 BOTANICAL GAZETTE [APRIL 


33. LEHMANN, E., Uber katalytische Lichtwirkung bei der Samenkeimung. 
Biochem. Zeitschr. 50:338-392. 1913. 

34. , Lichtkeimungsfragen. Zeits. fiir Bot. 7:560-580. 1015. 

35. LIEBENBERG, A., Uber den Einfluss des Lichtes intermittierender Erwarm- 
ung auf die Keimung von Samen. Bot. Centralbl. 18:21-26. 1884. 
36. LEHMANN, E., and OTTENWALDER, A., Uber katalytische Wirkung des 
Lichtes bei der Keimung lichtempfindlicher Samen. Zeits. fiir Bot. 

5: 337-364. 1913. 

37. Motiscu, H., Uber ein einfaches Verfahren Pflanzen zu treiben (Warmbad 
Methode). Sitzungber. Kaiserl. Akad. Wissensch.°117:87-117. 1908. 

38. NoOBBE, Fr., Handbuch der Samen Runde. 1876. 

30. , Ubt das Licht einen vorteilhaften Einfluss auf das Keimung des 
Grassamen. Land. Vers. Stat. 27:347-355. 1882. 

40. OTTENWALDER, A., Lichtintensitét und Substrat bei der Lichtkeimung. - 
Zeits. fiir Bot. 6:785-848. 1914. 

41. Paucuon, A., Recherches sur le réle de la lumiére dans la germin. Etude 
historique, critique et physiologique. Ann. Sci. Nat. Bot. VI. 10: 1880. 

42. PICKHOLTZ, L., Ein Beitrag zur Frage tiber die Wirkung des Lichtes und 
intermittierender Temperatur auf die Keimung von Samen, sowie iiber 
die Rolle des Wassergehaltes der Samen bei dieser Wirkung. Zeitschr. 
Landw. Versuch. Osterreich. 14:124-151. III. 

43. RACIBORSKI, W., Uber die Keimung der Tobaksamen. Bull. Inst. Bot. 
Buitenzorg. No. 6. 1900. 

44. REMER, W., Der Einfluss des Lichtes auf. die Keimung bei Phacelia tana- 
cetifolia Benth. Ber. Deutsch. Bot. Gesells. 22:328-329. 1906. 

45. Rostrup, O., Aarberetning fra Dansk Froekontroll. 1896; rev. Exper. 
Sta. Record 10:53-54. 1808. 

46. SHULL, C. A., Oxygen minimum and the germination of Xanthium seed. 
BOUrGAZ, 522453477. Loum: 

47. Stmon, Fr., Uber die Keimung zuvor belichteter und chemische vorbe- 
handelter Samen. Biochem. Zeits. 48:410-417. I9I3. 

48. STEBLER, E. G., Uber den Einfluss des Lichtes auf die Keimung. Bot. 
Centralbl. 7:157-158. 1881. 

49. TAammes, T., Uber den Einfluss der Sonnenstrahlen auf die Keimfahigkeit 
von Samen. Landw. Jahrb. 29:467-482. 1900. 

50. TopAro, F., Action of sulphuric acid upon seed, particularly upon the 
hard seeds of certain Leguminosae. Staz. Sper. Agr. Ital. 34:613-6809. 
TQOI. 

51. WIESNER, J., Viscum album. Denkschrift Wiener. Akad. Wissensch. 1878. 


- 
hes 
} 
J 
7 
3 
4 
y 
*? 
ae 
+ sod 
Pye 
Min 
‘ 
3 « 
é : 
ai ee" 
} 4 


4? ay re ad Ga Perens ; 
(4, i, bsg : - (tah Bee Eye ok ee Pate al 3 

; 3 Tae preeeynaeiny r Re iz tah: itty! Hi rk wie; gi 
« ean Ay Oler cen iTiis! Brsab dha, £ icthetin Pagioeyetie ig 
~aynd Ant a beh pis idan Sal hi bee Len tenho try ae 
yt. Sienna ed Oe aeiemedeeerd Ae @ sore, kagely cadkg. | Viste fe 


‘ ia 43 ae ot sig Pape ong “ pactifchie Th, saa Pi ae 
A, ee 
a boas _ Lah art ee re Sar i. ea . : 
2 Ng 9 Musee eg te bles: : » Pee i, ras, i 


— 


: og =~ ae F, , hs" 
in a ~ ‘ ‘ &, 16% ced Pe 4 Liar , 

‘ > 4 ke ied. } 7 —) ee oF 14 AL. 4 i 
aaa 4 7 : { ‘ 


nee, al ht dea LT ee on in ddae Gul ry 
oa ieee V4 bre va : is ah if oh as. ty et na : : * aL 4 AS ‘ony - 7 (Gy ar 

} * ax ‘: rr A wy u Lee L c fs 7 ‘ ii Hh ie “ert! - sibieds ar phe, Ce “ . 

{ he i - 34 ' ha ‘ "4 a M4 can, Ate ‘ ‘ 4 \ . ms ‘<4 
; - Pane i ina: Ee im Bit WoO ae / Uy Tiaatwe Mf pa rte al ¥ w 
aioriin ee a phy Odes) INR. AY MK. i Bat 
: a aed ae ' y it or . i } - 
t ay ; rf — Jere 1% Opa? } y yr 4 F 


jag el a : . 7 4 
~~ ACTBORI KE. 4 Jay. Cae ¥ ITM : 
> 69 é ry q , 
4 4 ¢ - ¥ * 
ai; i eee, oa CCl Wank et Ava: }.. i at. O .@ rite bet ‘ 
th Rl AR Shee, RP iyttach Ltod.. Jror gt. 34i59° a 


Po" pe lanier Ch iecerethtee Sen Danie Beeman een: Pa 


of ig 4 XS \* ” id 
| 28: ota oe Oa eee eae ra inn rain od a 
5 : iA t BW U5 tl, j 
OS ay Ota, 3 : Dhue fe Len geri ie ete | 


~ 


os ’ ic ee a4 ~~, ie PP ia) ie i eh e , 5g” a’ it2 se - X 1% 
i Tah ty Leer Tn 72 « tte | =e ’ nth 
hE CRO a” FA Abe al wi 4. Y ison ° aA OR ie 


oc i * ‘tet bey it fe tae hal naps ae, wet Nine I 


: Be F i, Sa 1 % , : ‘ a ~~ 7 
aie? 2 Waeingk-J. dine ian D en dint ho ag wiles. 7 

Mie oT Ss ; Le p é eG) 

| ‘ rs ey: Sy ey in ) oe Lay V6 4 : es ae 

1 Ae ied. oo a ha Sein] ua | a) ‘a 


LOT 


IN 


